Influenza A and B viruses cause a highly contagious respiratory disease in humans. Influenza A viruses, which have been isolated from a wide variety of avian and mammalian species, are responsible for pandemics that have caused high mortality rates. Avian H5N1 influenza A viruses, which have spread from Asia to Europe and Africa, are prime candidates for causing the next pandemic. The current antivirals against influenza A virus target either the M2 ion channel protein (amantadine/rimantadine) or the neuraminidase (NA) (oseltamivir/zamanivir). Many of the human isolates of H5N1 viruses are already resistant to amantadine, and H5N1 viruses that are partially, or completely, resistant to oseltamivir have recently been reported, highlighting the need for new antivirals. Our goal is to target a different, highly conserved influenza A virus-encoded protein, the NS1 protein, for drug development. The NS1 protein of influenza A virus will be designated as the NS1A protein to distinguish it from the NS1 protein encoded by influenza B virus, which will be designated as the NS1B protein. Influenza B viruses appear to infect only humans: We will target two functions of the NS1A protein that are crucial for virus replication: (1) double- stranded RNA (dsRNA)-binding by its N-terminal RNA-binding domain;and (2) binding by its effector domain of a cellular protein required for the 3'end processing of cellular pre-mRNAs (the 30-kDa subunit of the cleavage and polyadenylation factor, or CPSF30). The NS1B protein also has a dsRNA-binding function that we will target. We have already obtained strong evidence that these NS1 binding sites are valid new targets for drug development and have identified key features of their three-dimensional structure by X-ray crystallography and nuclear magnetic resonance (NMR) methods. We will now proceed to identify, assay and develop small organic ligand inhibitors for these three NS1 domains. This project is based on a strong and ongoing collaboration between virology efforts in the Krug laboratory at the University of Texas at Austin (UT-Austin) and structural biology efforts in the Montelione laboratory at Rutgers University. This effort will be augmented by the participation of Drs. Robertus, Ellington and Anslyn at UT-Austin and by the facilities of the Texas Institute for Drug and Diagnostics Development. In addition, the Paessler laboratory at the University of Texas Medical Branch will carry out animal experiments to test our inhibitory compounds.